Stephen W. Spaulding

Hormonal control of the phosphorylation of histones, HMG proteins and other nuclear proteins

Hormone-dependent phosphorylation and dephosphorylation of nuclear proteins may play an important part in regulating nuclear function and specific gene expression. Some progress has been made in identifying specific nuclear proteins whose phosphorylation is affected by specific hormones; however, relatively little is known about the regulatory mechanisms involved, or about the molecular consequences of increased or decreased phosphorylation. It is suspected--but not yet proved--that cAMP-dependent effects on transcription are mediated at least partly by increases in nuclear cAMP-dependent protein kinase (A-kinase) activity, and consequent increases in the phosphorylation of specific chromatin proteins. In several instances, increased phosphorylation has been found to precede or correlate with cAMP-mediated induction of specific gene products. Several chromatin proteins are susceptible to cAMP-dependent phosphorylation in vivo, including histones H1 and H3, the high mobility group protein HMG 14 (which is preferentially associated with actively transcribed chromatin), and at least three other basic nonhistone proteins. The A-kinase phosphorylation sites of the majority of H1, H3 and HMG 14 molecules in chromatin appear to be inaccessible to A-kinase in vivo; nothing is known about the factors determining their accessibility, which may be tightly regulated and may vary significantly from cell to cell and tissue to tissue. Many hormone-induced changes in nuclear protein phosphorylation may be cAMP-independent. cAMP-independent mechanisms could involve a variety of nuclear enzymes including, for example, cGMP-dependent, Ca2+/calmodulin-dependent, Ca2+/phospholipid-dependent and polyamine-dependent protein kinases. So far, however, there is little solid evidence in support of a role for any specific cAMP-independent protein kinase in mediating hormonally induced increases in the phosphorylation of specific, identified nuclear proteins.

HMG 14 and protamine enhance ligation of linear DNA to form linear multimers: Phosphorylation of HMG 14 at Ser 20 specifically inhibits intermolecular DNA ligation

HMG 14 and protamine can be used to enhance intermolecular ligation of low concentrations of linear DNA. Adding HMG 14 (50 moles per mole DNA) caused 50% of blunt-ended DNA to form predominantly dimers, and all cohesive-ended DNA to form multimers (greater than 6-mer) in response to T4 ligase. Protamine was maximally effective at 40:1, producing mostly dimers and trimers. Adding higher concentrations of HMG 14 did not affect the ligation pattern of cohesive-ended DNA, while higher concentrations of protamine inhibit the formation of multimers. Phosphorylation of HMG 14 at Ser 20 by Ca(++)-phospholipid dependent protein kinase abolished the ability of HMG 14 to stimulate intermolecular ligation, but did not substantially interfere with intramolecular ligation, or the binding of HMG 14 to linear or circular DNA as assessed by gel mobility. Thus Ser 20, which is located in the amino terminal DNA-binding domain of HMG 14, appears to modulate DNA-DNA interactions.

A one-step preparative method for separating SER 6-phosphorylated HMG 14 from unphosphorylated HMG 14 and in vitro phosphorylation reaction components

While clear evidence exists for the regulation of the phosphorylation of the very basic high mobility group (HMG) and histone chromatin proteins, the physiological role of their phosphorylation remains poorly understood. Elucidation of these roles has been difficult, in part, because of the inability to obtain sufficient quantities of purified phosphorylated derivatives. We have used Mono S cation-exchange chromatography to prepare milligram quantities of pure Ser 6-phosphorylated HMG 14 (Ser 6-PO4-HMG) from unphosphorylated Mono S-purified calf thymus HMG 14 following in vitro phosphorylation with cAMP-dependent protein kinase (A-kinase). In one step, this technique separates the phosphorylated derivative from A-kinase, ATP, unphosphorylated HMG 14, and a minor phosphorylated by-product which evidence suggests may be the previously reported Ser 6, 24-diphospho-HMG 14. Mono S chromatography also enhances the purity of calf thymus HMG 14 prepared by perchloric acid extraction, acetone and ethanol precipitations, and CM-Sephadex chromatography. In addition, it permits the detection of apparent microheterogenous forms of both unphosphorylated and Ser 6-PO4-HMG 14. The significant reductions in binding affinity resulting from the incorporation of phosphate groups into HMG 14 suggest that Mono S chromatography could have more general application in the isolation of phosphorylated derivatives of other basic proteins, including other chromatin-associated DNA-binding proteins which are known to undergo specific phosphorylation. It would especially be useful when the proteins and their phosphorylated derivatives bind more tightly to Mono S than the kinases used for their phosphorylation.

TSH-treatment of thyroid slices increases the amount of DNA released from nuclei by mild DNase-I digestion

Nuclei from TSH-treated and control thyroid slices were subjected to very limited digestion by DNase I, and then centrifuged at 1200×g. The amount of DNA released into supernatants was increased significantly by TSH when <0.1% to 3% of total DNA was rendered acid-soluble. This effect could be detected in buffers containing 2mM Mg++ (with chromatin condensed) or <0.05mM Mg++ (chromatin decondensed). Gel electrophoresis showed that the length of the majority of the DNA fragments in the supernatants (<0.1% of DNA acid-soluble) was greater than 4 kilobases; no TSH-dependent shift in size distribution was observed. We speculate that TSH may affect specific DNase I-hypersensitive sites in chromatin.

The Thyroid and Thyroid Hormones

Thyroxine (T 4 ) produced by the thyroid gland undergoes deiodination in cells, which can use it either to form the more active triiodothyronine (T 3 ) or the inactive reverse T 3 (rT 3 ). Thyroid hormones and their metabolites can bind to receptors on the cell surface, or be transported inside the cell, where they can bind to cytosolic proteins or proceed to the nucleus and bind to nuclear thyroid hormone receptors, to regulate the expression of many genes.

Hormonal Regulation of the EGF/Receptor System

The EGF/receptor system is involved in the development, maintenance and repair of various organs, particularly those involving ductal systems. Disruptions of the EGF/receptor system have been implicated in a variety of clinical disorders including cancer and aging. Some hormones alter the expression of EGF ligands, EGF receptors and of downstream factors that mediate EGF receptor action during development. These hormonal effects vary according to the species, organ and stage of development under study. Androgens alter EGF mRNA levels in several rodent tissues, yet no androgen-responsiveness is detectable within 7 kB upstream of the promoter of the EGF gene, which supports earlier evidence that androgens have post-transcriptional actions on EGF expression. AU-rich elements that can influence transcript stability and subcellular localization are present in the 3’ UTR of EGF mRNA. Androgens regulate EGF mRNA polyadenylation site usage, poly-A tail length and mRNA stability in the mouse submaxillary salivary gland (SMG) but not in the kidney, although androgens do regulate other genes in the kidney. Androgens also regulate the levels of several AU-rich RNA binding proteins known to affect the subcellular localization, translation and stability of AU-rich mRNAs. Androgen-dependent changes in the subcellular levels of these RNA binding proteins also indicate a causal connection with the androgen-dependent changes in EGF mRNA and its translation.